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Porcine Fc gamma RIIb sub-isoforms are generated by alternative splicing
Veterinary Immunology and Immunopathology 145 (2012) 386–394
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Research paper
Porcine Fc gamma RIIb sub-isoforms are generated by alternative splicing Pingan Xia 1 , Xiaoping Liu 1 , Yina Zhang, Erzhen Duan, Zhiyuan Zhang, Jing Chen, Chunlong Mu, Baoan Cui ∗ College of Animal Husbandry and Veterinary, Henan Agricultural University, Zhengzhou, China
a r t i c l e
i n f o
Article history: Received 6 June 2011 Received in revised form 13 December 2011 Accepted 15 December 2011 Keywords: Fc␥ receptor Fc␥RIIb Sub-isoforms Alternative splicing Pig
a b s t r a c t Receptors for the Fc portion of IgG (Fc␥Rs) are expressed on various leukocytes and they modulate both humoral and cell-mediated immune responses with different capacities for IgG binding and phagocytosis. Four different types of Fc␥Rs, Fc␥RI (CD64), Fc␥RII (CD32), Fc␥RIII (CD16) and Fc␥RIV, have been identified. There are three Fc␥RII isoforms (activating Fc␥RIIa and Fc␥RIIc, and inhibitory Fc␥RIIb) in humans, one isoform (inhibitory Fc␥RIIb) in mice, and two isoforms (inhibitory Fc␥RIIb and activating Fc␥RIIc) in cattle. Two alternativly spliced isoforms of Fc␥RIIb, b1 and b2, have been identified in humans, mice and cattle, however, only two porcine Fc␥RIIb transcripts have been reported. In this study, we report the identification of three new porcine Fc␥RIIb transcript and analyze the sequences of five porcine Fc␥RIIb transcript generated by alternative splicing. The porcine transcript 1 and porcine transcript 2 have a high homology and structural similarity with human b1 and b2, respectively, while there is only one alanine residue difference at the signal peptide region between porcine transcript 1 and transcript 4, as well as porcine transcript 2 and transcript 3. This is the first time that an alternativly spliced isoform of porcine transcript 5 is described in pigs rather than humans or other animals. All the five transcripts have the consensus sequence of an ITIM (ITYSLL) in their cytoplasmic tails. Analysis results indicate that the five transcripts serve as inhibitory receptors and are these sub-isoforms or alternativly spliced isoforms. Immunoglobulin-binding assays show that transcript 1, transcript 2, transcript 3 and transcript 4 have binding activity for IgG immune complexes, whereas transcripts 5 without domain 2 can not bind IgG-complexes. It is now clear that porcine Fc␥RIIb exists as five sub-isoforms at least. These sub-isoforms may individually modulate Fc␥RIIb-mediated immune responses in the porcine immune system. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Fc␥Rs, which recognise the Fc-fragment of IgG antibodies, are expressed on almost all hemopoietic originated
∗ Corresponding author at: College of Animal Husbandry and Veterinary, Henan Agricultural University, No. 95 Wenhua Road, Zhengzhou 450002, China. Tel.: +86 371 6355 8516. E-mail address: [email protected] (B. Cui). 1 These authors contributed equally to this work. 0165-2427/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2011.12.012
cells, such as monocytes and macrophages, and in nonhemopoietic cells, such as endothelial cells (Ravetch and Kinet, 1991; Mantzioris et al., 1993; Van de Winkel and Capel, 1993). Fc␥Rs provide an essential linkage between humoral and cellular immunity, including mediating phagocytosis of IgG-coated pathogens and antibodydependent enhancement (ADE), promoting activation of effector cells, and leading to inflammatory response and antibody-mediated cellular cytotoxicity (ADCC) (Daeron, 1997; Ravetch and Bolland, 2001; Tirado and Yoon, 2003; Nimmerjahn and Ravetch, 2008). Four different
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types of Fc␥Rs have been identified. Fc␥RI (CD64) is a high affinity receptor with three immunoglobulin-like extracellular domains for IgG or IgG immune complexes, while Fc␥RII (CD32) and Fc␥RIII (CD16) are low affinity receptors with two extracellular domains for IgG immune complexes. Fc␥RIV, which was identified recently in mice, is an intermediate affinity receptor with two extracellular domains and distinct IgG subclass specificity (Mechetina et al., 2002; Davis et al., 2002; Nimmerjahn et al., 2005; Nimmerjahn and Ravetch, 2006). Fc␥RII has three isoforms, both inhibitory (Fc␥RIIb) and activating type in humans (Fc␥RIIa and Fc␥RIIc) (Fossati et al., 2001), and in cattle (Fc␥RIIc: NM001109806). Fc␥RIIb, a 40 kDa monomeric glycoprotein, is expressed on almost all the immune cells except T cells and NK cells (Brooks et al., 1989; Minskoff et al., 1998; Lynch, 2000; Davis et al., 2005; Su et al., 2007). Fc␥RIIb downregulates phagocytosis by an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic tail (McKenzie and Schreiber, 1998; Muta et al., 1994). The ITIM motif has been shown to be necessary and sufficient to mediate the inhibition of the BCR (B-cell receptor)-generated calcium mobilization and cellular proliferation (Amigorena et al., 1992; Tzeng et al., 2005; Joshi et al., 2006). The ITIM tyrosine of Fc␥RIIb is phosphorylated by Src kinase to generate a docking site for the SH2 domain of SHIP-1, which is critical for the inhibitory function of Fc␥RIIb (Ravetch and Lanier, 2000; Coggeshall, 1998). The inhibitory property of Fc␥RIIb serves as maintaining peripheral tolerance by regulating the threshold of activation responses, and terminating the IgG-mediated stimulations (Ravetch and Bolland, 2001). The Fc␥R isoforms on various immune cells are generated by alternative splicing. The gene of human Fc␥RIIa (CD32A) is alternatively spliced into two transcripts, one encoding a membrane associated isoform and the other encoding a soluble isoform that lacks the transmembrane region (Astier et al., 1994). Two alternativly spliced isoforms of Fc␥RIIb, designated b1 and b2, are known in humans and animals. They differ structurally in the accretion of a short cytoplasmic exon in the b1 form and the length varying among the examined species (ranging from 15 to 47 amino acid residues). Longer b1 insertions are found in rodents such as mice and guinea pigs, while shorter sequences are noted in primates (including human) and livestock species (Ravetch et al., 1986; Brooks et al., 1989; Yamashita et al., 1993; Firth et al., 2010). Three isoforms of porcine Fc␥RIIIa, Fc␥RIIIa1, Fc␥RIIIa2 and Fc␥RIIIa3, are generated by alternative splicing (Jie et al., 2009). The first porcine Fc␥RIIb has also been cloned and characterized. The porcine Fc␥RIIb is a transmembrane glycoprotein composed of two Ig-like extracelluar domains, a transmembrane region and a cytoplasmic tail with an ITIM motif (Qiao et al., 2006). Recently, we cloned a new porcine Fc␥RIIb isoform (Fc␥RIIb1) (Xia et al., 2011). Here we report the identification of three new porcine Fc␥RIIb transcripts and analyze the sequences of five porcine Fc␥RIIb transcripts generated by alternative splicing.
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2. Materials and methods 2.1. Cells and antibodies COS-7 cells were maintained in Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal calf serum (FCS). Polyclonal antibody (pAb) specific for porcine Fc␥RIIb (ELISA assay antibody titre: 5120) was derived from mice immunized with extracellular region recombinant proteins of porcine Fc␥RIIb coupled with Freund’s Incomplete Adjuvant (Liu et al., 2010). Negative pAb was derived from normal mice. FITC-labelled goat anti-mouse IgG and horseradish peroxidase (HRP)-conjugated rabbit anti-pig IgG were purchased from Beijing Boisynthesis Biotechnology Corporation. Pig anti-chicken erythrocyte IgG was purified by standard method from the hyperimmune serum of a piglet vaccinated with chicken erythrocytes (HI assay antibody titre, 1:32). 2.2. Cloning gene and constructing expression plasmid A blood sample was collected from a 2-month-old pig from a commercial farm. PBL (Peripheral blood leucocytes) cells were obtained from the whole blood of pig collected in acid citrate dextrose anticoagulant. PAM (Pulmonary alveolar macrophage) cells were obtained from a healthy pig by bronchoalveolar lavage after necropsy. RNA was extracted using the TRIzol reagent (Invitrogen) from PBL cells or PAM cells according to the manufacturer’s instruction. RT-PCR was performed with Revert Aid TM First strand cDNA Synthesis kits (Promega Corporation) as described by the manufacturer. The cDNA was amplified by PCR using the specific primers for porcine Fc␥RIIb(5 -GTGATGGGGATCCCCTCGTT-3 /5 ATTTAAATGTGGTTCTGGTAATCTGAAG-3 ). The amplicon was cloned into pTG19-T and sequenced. Then, the amplified cDNA was subcloned into pcDNA3 vector to generate the recombinant expressing plasmids. 2.3. Sequence and structural analysis Nucleotide and amino acid sequence alignment was analyzed with DNAMAN software package (6.0.3.99, Homology analysis was http://www.lynnon.com). performed using BLAST at the National Center for Biotechnological Information (NCBI). The structure of the sequence was characterized using SMART (http://smart.emblheidelberg.de). This algorithm allowed identification of putative signal peptide, Ig-like domain and transmembrane region. Detailed analysis of the published genomic sequence for the region including the CD32 locus, obtained from Sus scrofa chromosome 4 genomic contig (NW-001886241.1), was carried out using the GenomeScan intron/exon prediction online software package (http://genes.mit.edu/genomescan.html) or performed manually. 2.4. Transfection for transient expression COS-7 cells in 6-well plates were cultured in DMEM containing 10% FCS at 37 ◦ C in a humidified 5% CO2 atmosphere
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for 24 h, and replaced with medium without serum for 2 h afterwards. The cells were transiently transfected with 4 g of the expression plasmid combined with Lipofectamine 2000 in DMEM for 24 h, and selected by DMEM containing G418 (400 g/mL). After two weeks, the transfected cells were analyzed by flow cytometry and immunoglobulinbinding assay.
in transcript 1 form. The transcript 5 lacked 85 amino acids of the extracellular region comprising EC2, as well as 19 amino acids of the cytoplasmic tail also lacking from transcript 2 (b2 configuration). The five transcripts all had the consensus sequence ITIM (ITYSLL) in cytoplasmic tail.
2.5. Flow cytometry analysis
3.2. FcRIIb transcripts are generated by alternative splicing mechanism
The transfected cells were harvested and washed with PBS containing 3% FCS (PBS/FCS), and then incubated for 1 h at 4 ◦ C with pAb specific for extracellular region of porcine Fc␥RIIb for 1 h at 4 ◦ C. After three washes with PBS/FCS, the cells were incubated with FITC-labelled goat anti-mouse IgG for 2 h at 4 ◦ C, and washed thoroughly. Stained samples were analyzed by Epics Elite flow cytometer (Coulter Corp., Miami, FL). Background FITC fluorescence was determined by surface staining of cells with normal mouse sera and FITC-labelled goat anti-mouse IgG. 2.6. Immunoglobulin-binding assay Chicken erythrocytes were sensitized with pig antichicken erythrocyte IgG and were resuspended in serumfree medium. IgG-sensitized chicken erythrocytes were added to the transfected cells. After 45–60 min incubation at room temperature with occasional gentle agitation, the non-adherent erythrocytes were washed off with PBS. The monolayer cells were fixed with methanol for 10 min and stained with HRP-conjugated rabbit anti-pig IgG, then washed and incubated with 3-amino-9-ethylcarbazole (AEC) (Sigma). Stained samples were analyzed by microscope.
To determine whether five transcripts are generated by alternative splicing, the genomic sequence of porcine Fc␥RIIb (CU468575) was analyzed using the GenomeScan intron/exon prediction online software package or performed manually. Analysis results showed that genomic sequence of porcine Fc␥RIIb was composed of 8 introns and 9 exons (Fig. 2). Sizes of exon1, exon2, exon3, exon4, exon5, exon6, exon7, exon8 and exon9 were 112, 21, 258, 255, 255, 120, 57, 38 and 90 bp, respectively. Interestingly, the sequences of exon3 and exon4 were different only in adding 3 bp to the 5 end of exon3, resulting in the deletion of one amino acid and alteration of the affected second amino acid (Fig. 1). The cloned five transcripts all were generated exactly from the predicted exons of porcine Fc␥RIIb genomic sequence by alternative splicing style, the first two nucleotides of the consensus splicing donor (GT) and acceptor (AG) (Fig. 3). The transcript 1 was composed of exons1–3, 5–9; The transcript 2 was composed of exons1, 2, 4–6, 8, 9; The transcript 3 was composed of exons1–3, 5, 6, 8, 9; The transcript 4 was composed of exons1, 2, 4–9; The transcript 5 was composed of exons1–4, 6, 8, 9.
3. Results
3.3. Construction of FcRIIb expression plasmids
3.1. Sequence and structural analysis
The cDNAs of porcine Fc␥RIIb transcript 1–5 were cloned into pcDNA3 vector to generate the expression plasmids pcDNA-b1, pcDNA-b2, pcDNA-b3, pcDNA-b4, pcDNA-b5, respectively. The PCR-generated plasmids were verified by sequencing of the entire insert.
To determine whether Fc␥RIIb exists in multiple isoforms in pigs, RT-PCR was performed using a pair of primers designed by the published porcine Fc␥RIIb sequence (DQ026064). The five Fc␥RIIb transcripts were cloned from porcine PBL cells or PAM cells and sequenced respectively. The sequence analysis revealed that sizes of transcript 1 (FJ608551), transcript 2 (HM803103), transcript 3 (HM803104), transcript 4 (GQ994431) and transcript 5 (NM122239) were 951, 891, 894, 948 and 639 bp, respectively. The homology of nucleotide sequence between five transcripts was 97.2–99.4%. The predicted amino acid sequences of five transcripts were shown in Fig. 1. The structural analysis revealed that transcript 1 was composed of an N-terminal secretory signal peptide (1-45aa), an extracellular region including domain 1 (EC1) and domain 2 (EC2) (46-224aa), a putative transmembrane domain (225–247aa) and a cytoplasmic tail (248–316aa). The transcript 2 was lacking an alanine residue of signal peptide and a 19 amino acid insert of cytoplasmic tail in transcript 1 form. The transcript 3 lacked a 19 amino acid insert of cytoplasmic tail in transcript 1 form. The transcript 4 was short of an alanine residue of signal peptide
3.4. Expression of the porcine FcRIIb on the surface of COS-7 cells To determine whether five porcine Fc␥RIIb transcripts can be expressed on the surface of the cells, COS-7 cells were transfected with pcDNA-b1, or pcDNA-b2, or pcDNAb3, or pcDNA-b4, or pcDNA-b5, and then were respectively selected by G418 (400 g/mL). The obtained transfected cells were stained with specific pAb for extracellular region of porcine Fc␥RIIb and FITC-labelled goat anti-mouse IgG, and then analyzed by flow cytometry. The median fluorescence intensities (MFI) value (the positive population) of the transfected cells surface-stained with Fc␥RIIb-specific pAb was significantly increased compared with the transfected cells stained with Fc␥RIIb-negative pAb (Fig. 4), indicating that five transcripts all were expressed on the cell surface.
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Fig. 1. Comparation of amino acid sequence of porcine Fc␥RIIb transcripts. The signal peptide is marked by orange shade. The extracellular domain 1(EC1) and extracellular domain 2(EC2) are marked by blue shade. The transmembrane region is marked by green shade. The immunoreceptor tyrosine-based inhibitory motif (ITIM) is marked by crimson shade. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
3.5. Function of FcRIIb transcripts binding IgG immune complexes To test whether the porcine Fc␥RIIb can bind IgG immune complexes, IgG-sensitized chicken erythrocytes were used in binding assay with Fc␥RIIb transfected cells. Results showed that COS-7 cells transfected respectively with pcDNA-b1, pcDNA-b2, pcDNA-b3 and pcDNA-b4 were able to bind IgG-sensitized chicken erythrocytes. However, COS-7 cells transfected respectively with pcDNA-b5 and pcDNA3 showed no ability to bind IgG-sensitized erythrocytes, and the non-transfected COS-7 cells did not bind IgG-sensitized erythrocytes (Fig. 5). The binding assay
further confirmed the function of the porcine Fc␥RIIb transcript 1–4 binding IgG immune complexes and identified them as putative IgG receptors. 4. Discussion We have isolated and characterized full-length cDNAs encoding five porcine Fc␥RIIb transcripts, of which three are cloned and characterized for the first time, except the previously identified transcript 3 and transcript 1 (Qiao et al., 2006; Xia et al., 2011). Some problems remain in selecting the appropriate isoforms to make comparisons, since the naming convention of the receptor is based on the
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Fig. 2. Schematic representation of porcine Fc␥RIIb genomic structure. The exon sequence of porcine Fc␥RIIb is showed. The five cloned porcine Fc␥RIIb transcripts all are generated exactly from the predicted exons of porcine Fc␥RIIb genomic sequence by alternative splicing style, the first two nucleotides of the consensus splicing donors (GT) and acceptor (AG).
time order of new discovery rather than sequence homology or structure. Here we take published human Fc␥RIIb sequences as reference for our newly discovered porcine Fc␥RIIb transcripts. In humans, two spliced isoforms of Fc␥RIIb, b1 and b2, are distinguished by the inclusion of a 19 amino acid in-frame insertion in the cytoplasmic tail
of b1 (Amigorena et al., 1992; Brooks et al., 1989; Firth et al., 2010). BLAST analysis shows that porcine transcript 1 is similar to human b1 and shares 77.4% homology with amino acid sequence of human b2, while porcine transcript 2 is the homologue of human b2, and shares 65.4% homology with amino acid sequence of human b2. The same as
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Fig. 3. Schematic representation of cDNA structure of five porcine Fc␥RIIb transcripts, which made up the different exons.
their human homologues, porcine transcript 1 also has a 19 amino acid in-frame insertion in the cytoplasmic domain whereas transcript 2 does not. It indicates that porcine transcript 1 may be recognised as the porcine Fc␥RIIb1 isoform, and transcript 2 as the porcine Fc␥RIIb2 isoform. There is only one alanine residue difference in the signal peptide region between porcine transcript 1 and transcript 4, or between transcript 2 and transcript 3. Moreover, transcript 3 and transcript 4 are also generated precisely by splicing style, the first two nucleotides of the consensus splicing donor (GT) and acceptor (AG), indicating that porcine transcript 3 or transcript 4 should belong to two new porcine Fc␥RIIb isoforms rather than mutants of transcript 2 and transcript 1. The identification of spliced form of the porcine transcript 5 is of particular interest, because this is the first time for the spliced form to be described in pigs rather than other animals and humans. This indicates that porcine transcript 5 is also a new novel porcine Fc␥RIIb isoform. It is clear that porcine Fc␥RIIb has at least five sub-isoforms now. The complexity of Fc␥RIIb-mediated immune response is demonstrated by the existence of Fc␥RIIb isoforms generated by alternative splicing. It is clear from earlier studies that both the murine and human b2 proteins undergo endocytosis upon binding ligand (Miettinen et al., 1989, 1992; Budde et al., 1994a; Van Den Herik-Oudijk et al., 1994). However, the endocytosis of immune complexs (ICs) mediated by b2 is not same as b1 in mice (Miettinen et al., 1989, 1992; Budde et al., 1994a). The b1 form uniquely prevents uptake of antigen-antibody complexes via clathrin-coated pits (Miettinen et al., 1992). It can be attributable to the increased affinity of the b1 insertion for cytoskeletal components, resulting from the association of b1 proteins with actin molecules and membrane phospholipids (Miettinen et al. 1992; Chen et al., 1999). Since immune complexes bound to b1 cannot be processed through the endocytic pathway, clonotypic expansion of Ag-specific T cells is prevented due to the lack of MHCII peptide presentation (Amigorena et al., 1992; Minskoff et al., 1998). In humans, bl and b2 display no notable difference in their signaling capabilities, as they both undergo
tyrosine phosphorylation, enhancing SHIP phosphorylation and downregulating cellular endocytosis (Joshi et al., 2006). However, there are conflicting reports with regard to the ability of b2 to become tyrosine-phosphorylated. In B cells, the human b2 ITIM tyrosine is not phosphorylated although its ability to downregulate antigen receptorinduced calcium mobilization is comparable to that of bl, which is tyrosine-phosphorylated (Budde et al., 1994b). Although the molecular details of this paradoxical observation are not fully understood, it is clear that human bl and b2 likely serve some non-overlapping functions in B cells. Of note, there are no studies to date comparing the functional capacity of porcine Fc␥RIIbl and b2. Likewise, it is not known whether both porcine Fc␥RIIbl and Fc␥RIIb2 are tyrosine-phosphorylated in B cells, macrophages and monocytes, and whether they function as inhibitory receptors during phagocytosis. In addition, in order to examine the tissue distribution of the porcine Fc␥RIIb in vivo, we surveyed amplicons by RT-PCR from porcine PBL cells and different tissue samples (muscle, thymus, lung, kidney, inguinal lymph node, spleen, tonsil, heart, liver) obtained from individual animals of a variety of porcine breeds, using the same primers as for PAM cells. The results showed that the porcine transcript 1 and transcript 3 were both detected and exhibited predominant expression in these tissues, PBL cells and PAM cells (data not shown). While the detection of these spliced variants is of great interest, it is beyond the scope of current research to elucidate mechanisms of the predominant expression pattern and the precise biological function. This topic is certainly a key area for future examination. Soluble Fc␥RIIb is first characterized in mice (Fridman and Golstein, 1974; Newport-Sautes et al., 1975). Soluble Fc␥RIIb is generated either by the cleavage of membranebound protein, or as soluble spliced variants lacking the exon coding for the transmembrane (TM) region (Fridman et al., 1993; Tartour et al., 1993; Galon et al., 1997). Membrane-cleaved soluble FcR can be produced by virtually any cell expressing the full-length protein, but it seems to be mainly produced by lymphocytes, at least in mice (Fridman et al., 1993). The TM negative form,
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Fig. 4. Flow cytometry analysis of expression of porcine Fc␥RIIb on the surface of COS-7 cells. (1) Representative histograms of the median fluorescence intensities (the positive population) of pcDNA-b1 transfected COS-7 cells surface-stained with Fc␥RIIb-specific pAb(A) or Fc␥RIIb-negative pAb(B). (2) The comparison of the MFI values of transfected COS-7 cells after surface staining with Fc␥RIIb-specific pAb and Fc␥RIIb-negative pAb. The data are means ± S.D. of three experiments, which are respectively 25.3 ±0.1 (A); 2.7 ± 0.2 (B); 17.7 ± 0.1 (C); 2.0 ± 0.2 (D); 20.0 ± 0.1 (E); 2.2 ± 0.2(F); 18.3 ±0.1 (G); 1.3 ± 0.1 (H); 14.4 ± 0.2 (I); 1.5 ±0.1 (J); 2.3 ± 0.2 (K); 1.5 ± 0.1 (L); 2.5 ± 0.1 (M); and 1.3 ± 0.1 (N).
however, is typically produced only by cells capable of expressing Fc␥RIIb2, such as T cells, macrophages and Langerhans. It is present in higher serum concentrations than the membrane-cleaved version (Fridman et al., 1993; Tartour et al., 1993). In cattle, Fc␥RIIb3, a soluble Fc␥RIIb, is characterized by the absence of a 40 amino acid region
concluding the predicted TM domain in b1 form. This transcript appears to have much more limited expression profile, which may indicate that the expression varies with the cellular activation-state (Firth et al., 2010). However, the transcript similar to bovine Fc␥RIIb3 variant has not been isolated in pig yet.
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Fig. 5. COS-7 cells transfected with porcine Fc␥RIIb genes bind IgG-sensitized erythrocytes (A) COS-7 cells transfected with pcDNA-b1. (B) COS-7 cells transfected with pcDNA-b2. (C) COS-7 cells transfected with pcDNA-b3. (D) COS-7 cells transfected with pcDNA-b4. (E) COS-7 cells transfected with pcDNA-b5. (F) COS-7 cells transfected with pcDNA3. (G) COS-7 cells.
The porcine alternatively spliced transcript 5 is particularly interesting. It is the first time that this form has been described in pigs rather than other animals and humans. Our results show the transcript 5 is unable to interact with immune complexes, despite its expression on the cell surface. One possible reason is that this protein lacks the EC2 domain, which is responsible for interacting with immune complexes and subsequent activations of innate immune cells (Hulett and Hogarth, 1994; Hulett et al., 1994, 1995). However, previous study reported that the EC1 domain of one Fc␥RIII receptor interacted with the EC2 domain of a successive Fc␥RIII molecule, which increased the accessibility of Fc␥RIII to the Fc portion of immune complexes and efficiently activated Fc␥RIII-inducing signals (Zhang et al., 2000). The function of transcript 5 in the porcine Fc␥RIIb-mediated immune responses needs further investigation. Generally, we have cloned five porcine Fc␥RIIb transcripts that are apparently generated according to the predicted splicing style. It is now clear that porcine Fc␥RIIb exists as at least five sub-isoforms, four of which can bind IgG. These isoforms may respectively modulate Fc␥RIIb-mediated immune responses in pigs. Hence, further functional characterization of Fc␥RIIb isoforms will provide new insight into better understanding of the
mechanism how these isoforms regulate the Fc␥RIIbmediated immune responses in porcine system. Acknowledgments We thank Li Hongwei and Chen Liying for reviewing the manuscript. This work was supported by National Natural Science Foundation of China (31172346). References Amigorena, S., Bonnerot, C., Drake, J.R., Choquet, D., Hunziker, W., Guillet, J.G., Webster, P., Sautes, C., Mellman, I., Fridman, W.H., 1992. Cytoplasmic domain heterogeneity and functions of IgG Fc receptors in B lymphocytes. Science 256, 1808–1812. Astier, A., de la Salle, H., de la Salle, C., Bieber, T., Esposito-Farese, M.E., Freund, M., Cazenave, J.P., Fridman, W.H., Teillaud, J.L., Hanau, D., 1994. Human epidermal Langerhans cells secrete a soluble receptor for IgG (Fc gamma RII/CD32) that inhibits the binding of immune complexes to Fc gamma R+ cells. J. Immunol. 152, 201–212. Brooks, D.G., Qui, W.Q., Luster, A.D., Ravetch, J.V., 1989. Structure and expression of human IgG FcRII (CD32), Functional heterogeneity is encoded by the alternatively spliced products of multiple genes. J. Exp. Med. 170, 1369–1385. Budde, P., Bewarder, N., Weinrich, V., Schulzeck, O., Frey, J., 1994a. Tyrosine-containing sequence motifs of the human immunoglobulin G receptors FcRIIb1 and FcRIIb2 essential for endocytosis and regulation of calcium flux in B cells. J. Biol. Chem. 269, 30636–30644.
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Research paper
Porcine Fc gamma RIIb sub-isoforms are generated by alternative splicing Pingan Xia 1 , Xiaoping Liu 1 , Yina Zhang, Erzhen Duan, Zhiyuan Zhang, Jing Chen, Chunlong Mu, Baoan Cui ∗ College of Animal Husbandry and Veterinary, Henan Agricultural University, Zhengzhou, China
a r t i c l e
i n f o
Article history: Received 6 June 2011 Received in revised form 13 December 2011 Accepted 15 December 2011 Keywords: Fc␥ receptor Fc␥RIIb Sub-isoforms Alternative splicing Pig
a b s t r a c t Receptors for the Fc portion of IgG (Fc␥Rs) are expressed on various leukocytes and they modulate both humoral and cell-mediated immune responses with different capacities for IgG binding and phagocytosis. Four different types of Fc␥Rs, Fc␥RI (CD64), Fc␥RII (CD32), Fc␥RIII (CD16) and Fc␥RIV, have been identified. There are three Fc␥RII isoforms (activating Fc␥RIIa and Fc␥RIIc, and inhibitory Fc␥RIIb) in humans, one isoform (inhibitory Fc␥RIIb) in mice, and two isoforms (inhibitory Fc␥RIIb and activating Fc␥RIIc) in cattle. Two alternativly spliced isoforms of Fc␥RIIb, b1 and b2, have been identified in humans, mice and cattle, however, only two porcine Fc␥RIIb transcripts have been reported. In this study, we report the identification of three new porcine Fc␥RIIb transcript and analyze the sequences of five porcine Fc␥RIIb transcript generated by alternative splicing. The porcine transcript 1 and porcine transcript 2 have a high homology and structural similarity with human b1 and b2, respectively, while there is only one alanine residue difference at the signal peptide region between porcine transcript 1 and transcript 4, as well as porcine transcript 2 and transcript 3. This is the first time that an alternativly spliced isoform of porcine transcript 5 is described in pigs rather than humans or other animals. All the five transcripts have the consensus sequence of an ITIM (ITYSLL) in their cytoplasmic tails. Analysis results indicate that the five transcripts serve as inhibitory receptors and are these sub-isoforms or alternativly spliced isoforms. Immunoglobulin-binding assays show that transcript 1, transcript 2, transcript 3 and transcript 4 have binding activity for IgG immune complexes, whereas transcripts 5 without domain 2 can not bind IgG-complexes. It is now clear that porcine Fc␥RIIb exists as five sub-isoforms at least. These sub-isoforms may individually modulate Fc␥RIIb-mediated immune responses in the porcine immune system. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Fc␥Rs, which recognise the Fc-fragment of IgG antibodies, are expressed on almost all hemopoietic originated
∗ Corresponding author at: College of Animal Husbandry and Veterinary, Henan Agricultural University, No. 95 Wenhua Road, Zhengzhou 450002, China. Tel.: +86 371 6355 8516. E-mail address: [email protected] (B. Cui). 1 These authors contributed equally to this work. 0165-2427/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2011.12.012
cells, such as monocytes and macrophages, and in nonhemopoietic cells, such as endothelial cells (Ravetch and Kinet, 1991; Mantzioris et al., 1993; Van de Winkel and Capel, 1993). Fc␥Rs provide an essential linkage between humoral and cellular immunity, including mediating phagocytosis of IgG-coated pathogens and antibodydependent enhancement (ADE), promoting activation of effector cells, and leading to inflammatory response and antibody-mediated cellular cytotoxicity (ADCC) (Daeron, 1997; Ravetch and Bolland, 2001; Tirado and Yoon, 2003; Nimmerjahn and Ravetch, 2008). Four different
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types of Fc␥Rs have been identified. Fc␥RI (CD64) is a high affinity receptor with three immunoglobulin-like extracellular domains for IgG or IgG immune complexes, while Fc␥RII (CD32) and Fc␥RIII (CD16) are low affinity receptors with two extracellular domains for IgG immune complexes. Fc␥RIV, which was identified recently in mice, is an intermediate affinity receptor with two extracellular domains and distinct IgG subclass specificity (Mechetina et al., 2002; Davis et al., 2002; Nimmerjahn et al., 2005; Nimmerjahn and Ravetch, 2006). Fc␥RII has three isoforms, both inhibitory (Fc␥RIIb) and activating type in humans (Fc␥RIIa and Fc␥RIIc) (Fossati et al., 2001), and in cattle (Fc␥RIIc: NM001109806). Fc␥RIIb, a 40 kDa monomeric glycoprotein, is expressed on almost all the immune cells except T cells and NK cells (Brooks et al., 1989; Minskoff et al., 1998; Lynch, 2000; Davis et al., 2005; Su et al., 2007). Fc␥RIIb downregulates phagocytosis by an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic tail (McKenzie and Schreiber, 1998; Muta et al., 1994). The ITIM motif has been shown to be necessary and sufficient to mediate the inhibition of the BCR (B-cell receptor)-generated calcium mobilization and cellular proliferation (Amigorena et al., 1992; Tzeng et al., 2005; Joshi et al., 2006). The ITIM tyrosine of Fc␥RIIb is phosphorylated by Src kinase to generate a docking site for the SH2 domain of SHIP-1, which is critical for the inhibitory function of Fc␥RIIb (Ravetch and Lanier, 2000; Coggeshall, 1998). The inhibitory property of Fc␥RIIb serves as maintaining peripheral tolerance by regulating the threshold of activation responses, and terminating the IgG-mediated stimulations (Ravetch and Bolland, 2001). The Fc␥R isoforms on various immune cells are generated by alternative splicing. The gene of human Fc␥RIIa (CD32A) is alternatively spliced into two transcripts, one encoding a membrane associated isoform and the other encoding a soluble isoform that lacks the transmembrane region (Astier et al., 1994). Two alternativly spliced isoforms of Fc␥RIIb, designated b1 and b2, are known in humans and animals. They differ structurally in the accretion of a short cytoplasmic exon in the b1 form and the length varying among the examined species (ranging from 15 to 47 amino acid residues). Longer b1 insertions are found in rodents such as mice and guinea pigs, while shorter sequences are noted in primates (including human) and livestock species (Ravetch et al., 1986; Brooks et al., 1989; Yamashita et al., 1993; Firth et al., 2010). Three isoforms of porcine Fc␥RIIIa, Fc␥RIIIa1, Fc␥RIIIa2 and Fc␥RIIIa3, are generated by alternative splicing (Jie et al., 2009). The first porcine Fc␥RIIb has also been cloned and characterized. The porcine Fc␥RIIb is a transmembrane glycoprotein composed of two Ig-like extracelluar domains, a transmembrane region and a cytoplasmic tail with an ITIM motif (Qiao et al., 2006). Recently, we cloned a new porcine Fc␥RIIb isoform (Fc␥RIIb1) (Xia et al., 2011). Here we report the identification of three new porcine Fc␥RIIb transcripts and analyze the sequences of five porcine Fc␥RIIb transcripts generated by alternative splicing.
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2. Materials and methods 2.1. Cells and antibodies COS-7 cells were maintained in Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal calf serum (FCS). Polyclonal antibody (pAb) specific for porcine Fc␥RIIb (ELISA assay antibody titre: 5120) was derived from mice immunized with extracellular region recombinant proteins of porcine Fc␥RIIb coupled with Freund’s Incomplete Adjuvant (Liu et al., 2010). Negative pAb was derived from normal mice. FITC-labelled goat anti-mouse IgG and horseradish peroxidase (HRP)-conjugated rabbit anti-pig IgG were purchased from Beijing Boisynthesis Biotechnology Corporation. Pig anti-chicken erythrocyte IgG was purified by standard method from the hyperimmune serum of a piglet vaccinated with chicken erythrocytes (HI assay antibody titre, 1:32). 2.2. Cloning gene and constructing expression plasmid A blood sample was collected from a 2-month-old pig from a commercial farm. PBL (Peripheral blood leucocytes) cells were obtained from the whole blood of pig collected in acid citrate dextrose anticoagulant. PAM (Pulmonary alveolar macrophage) cells were obtained from a healthy pig by bronchoalveolar lavage after necropsy. RNA was extracted using the TRIzol reagent (Invitrogen) from PBL cells or PAM cells according to the manufacturer’s instruction. RT-PCR was performed with Revert Aid TM First strand cDNA Synthesis kits (Promega Corporation) as described by the manufacturer. The cDNA was amplified by PCR using the specific primers for porcine Fc␥RIIb(5 -GTGATGGGGATCCCCTCGTT-3 /5 ATTTAAATGTGGTTCTGGTAATCTGAAG-3 ). The amplicon was cloned into pTG19-T and sequenced. Then, the amplified cDNA was subcloned into pcDNA3 vector to generate the recombinant expressing plasmids. 2.3. Sequence and structural analysis Nucleotide and amino acid sequence alignment was analyzed with DNAMAN software package (6.0.3.99, Homology analysis was http://www.lynnon.com). performed using BLAST at the National Center for Biotechnological Information (NCBI). The structure of the sequence was characterized using SMART (http://smart.emblheidelberg.de). This algorithm allowed identification of putative signal peptide, Ig-like domain and transmembrane region. Detailed analysis of the published genomic sequence for the region including the CD32 locus, obtained from Sus scrofa chromosome 4 genomic contig (NW-001886241.1), was carried out using the GenomeScan intron/exon prediction online software package (http://genes.mit.edu/genomescan.html) or performed manually. 2.4. Transfection for transient expression COS-7 cells in 6-well plates were cultured in DMEM containing 10% FCS at 37 ◦ C in a humidified 5% CO2 atmosphere
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for 24 h, and replaced with medium without serum for 2 h afterwards. The cells were transiently transfected with 4 g of the expression plasmid combined with Lipofectamine 2000 in DMEM for 24 h, and selected by DMEM containing G418 (400 g/mL). After two weeks, the transfected cells were analyzed by flow cytometry and immunoglobulinbinding assay.
in transcript 1 form. The transcript 5 lacked 85 amino acids of the extracellular region comprising EC2, as well as 19 amino acids of the cytoplasmic tail also lacking from transcript 2 (b2 configuration). The five transcripts all had the consensus sequence ITIM (ITYSLL) in cytoplasmic tail.
2.5. Flow cytometry analysis
3.2. FcRIIb transcripts are generated by alternative splicing mechanism
The transfected cells were harvested and washed with PBS containing 3% FCS (PBS/FCS), and then incubated for 1 h at 4 ◦ C with pAb specific for extracellular region of porcine Fc␥RIIb for 1 h at 4 ◦ C. After three washes with PBS/FCS, the cells were incubated with FITC-labelled goat anti-mouse IgG for 2 h at 4 ◦ C, and washed thoroughly. Stained samples were analyzed by Epics Elite flow cytometer (Coulter Corp., Miami, FL). Background FITC fluorescence was determined by surface staining of cells with normal mouse sera and FITC-labelled goat anti-mouse IgG. 2.6. Immunoglobulin-binding assay Chicken erythrocytes were sensitized with pig antichicken erythrocyte IgG and were resuspended in serumfree medium. IgG-sensitized chicken erythrocytes were added to the transfected cells. After 45–60 min incubation at room temperature with occasional gentle agitation, the non-adherent erythrocytes were washed off with PBS. The monolayer cells were fixed with methanol for 10 min and stained with HRP-conjugated rabbit anti-pig IgG, then washed and incubated with 3-amino-9-ethylcarbazole (AEC) (Sigma). Stained samples were analyzed by microscope.
To determine whether five transcripts are generated by alternative splicing, the genomic sequence of porcine Fc␥RIIb (CU468575) was analyzed using the GenomeScan intron/exon prediction online software package or performed manually. Analysis results showed that genomic sequence of porcine Fc␥RIIb was composed of 8 introns and 9 exons (Fig. 2). Sizes of exon1, exon2, exon3, exon4, exon5, exon6, exon7, exon8 and exon9 were 112, 21, 258, 255, 255, 120, 57, 38 and 90 bp, respectively. Interestingly, the sequences of exon3 and exon4 were different only in adding 3 bp to the 5 end of exon3, resulting in the deletion of one amino acid and alteration of the affected second amino acid (Fig. 1). The cloned five transcripts all were generated exactly from the predicted exons of porcine Fc␥RIIb genomic sequence by alternative splicing style, the first two nucleotides of the consensus splicing donor (GT) and acceptor (AG) (Fig. 3). The transcript 1 was composed of exons1–3, 5–9; The transcript 2 was composed of exons1, 2, 4–6, 8, 9; The transcript 3 was composed of exons1–3, 5, 6, 8, 9; The transcript 4 was composed of exons1, 2, 4–9; The transcript 5 was composed of exons1–4, 6, 8, 9.
3. Results
3.3. Construction of FcRIIb expression plasmids
3.1. Sequence and structural analysis
The cDNAs of porcine Fc␥RIIb transcript 1–5 were cloned into pcDNA3 vector to generate the expression plasmids pcDNA-b1, pcDNA-b2, pcDNA-b3, pcDNA-b4, pcDNA-b5, respectively. The PCR-generated plasmids were verified by sequencing of the entire insert.
To determine whether Fc␥RIIb exists in multiple isoforms in pigs, RT-PCR was performed using a pair of primers designed by the published porcine Fc␥RIIb sequence (DQ026064). The five Fc␥RIIb transcripts were cloned from porcine PBL cells or PAM cells and sequenced respectively. The sequence analysis revealed that sizes of transcript 1 (FJ608551), transcript 2 (HM803103), transcript 3 (HM803104), transcript 4 (GQ994431) and transcript 5 (NM122239) were 951, 891, 894, 948 and 639 bp, respectively. The homology of nucleotide sequence between five transcripts was 97.2–99.4%. The predicted amino acid sequences of five transcripts were shown in Fig. 1. The structural analysis revealed that transcript 1 was composed of an N-terminal secretory signal peptide (1-45aa), an extracellular region including domain 1 (EC1) and domain 2 (EC2) (46-224aa), a putative transmembrane domain (225–247aa) and a cytoplasmic tail (248–316aa). The transcript 2 was lacking an alanine residue of signal peptide and a 19 amino acid insert of cytoplasmic tail in transcript 1 form. The transcript 3 lacked a 19 amino acid insert of cytoplasmic tail in transcript 1 form. The transcript 4 was short of an alanine residue of signal peptide
3.4. Expression of the porcine FcRIIb on the surface of COS-7 cells To determine whether five porcine Fc␥RIIb transcripts can be expressed on the surface of the cells, COS-7 cells were transfected with pcDNA-b1, or pcDNA-b2, or pcDNAb3, or pcDNA-b4, or pcDNA-b5, and then were respectively selected by G418 (400 g/mL). The obtained transfected cells were stained with specific pAb for extracellular region of porcine Fc␥RIIb and FITC-labelled goat anti-mouse IgG, and then analyzed by flow cytometry. The median fluorescence intensities (MFI) value (the positive population) of the transfected cells surface-stained with Fc␥RIIb-specific pAb was significantly increased compared with the transfected cells stained with Fc␥RIIb-negative pAb (Fig. 4), indicating that five transcripts all were expressed on the cell surface.
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Fig. 1. Comparation of amino acid sequence of porcine Fc␥RIIb transcripts. The signal peptide is marked by orange shade. The extracellular domain 1(EC1) and extracellular domain 2(EC2) are marked by blue shade. The transmembrane region is marked by green shade. The immunoreceptor tyrosine-based inhibitory motif (ITIM) is marked by crimson shade. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
3.5. Function of FcRIIb transcripts binding IgG immune complexes To test whether the porcine Fc␥RIIb can bind IgG immune complexes, IgG-sensitized chicken erythrocytes were used in binding assay with Fc␥RIIb transfected cells. Results showed that COS-7 cells transfected respectively with pcDNA-b1, pcDNA-b2, pcDNA-b3 and pcDNA-b4 were able to bind IgG-sensitized chicken erythrocytes. However, COS-7 cells transfected respectively with pcDNA-b5 and pcDNA3 showed no ability to bind IgG-sensitized erythrocytes, and the non-transfected COS-7 cells did not bind IgG-sensitized erythrocytes (Fig. 5). The binding assay
further confirmed the function of the porcine Fc␥RIIb transcript 1–4 binding IgG immune complexes and identified them as putative IgG receptors. 4. Discussion We have isolated and characterized full-length cDNAs encoding five porcine Fc␥RIIb transcripts, of which three are cloned and characterized for the first time, except the previously identified transcript 3 and transcript 1 (Qiao et al., 2006; Xia et al., 2011). Some problems remain in selecting the appropriate isoforms to make comparisons, since the naming convention of the receptor is based on the
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Fig. 2. Schematic representation of porcine Fc␥RIIb genomic structure. The exon sequence of porcine Fc␥RIIb is showed. The five cloned porcine Fc␥RIIb transcripts all are generated exactly from the predicted exons of porcine Fc␥RIIb genomic sequence by alternative splicing style, the first two nucleotides of the consensus splicing donors (GT) and acceptor (AG).
time order of new discovery rather than sequence homology or structure. Here we take published human Fc␥RIIb sequences as reference for our newly discovered porcine Fc␥RIIb transcripts. In humans, two spliced isoforms of Fc␥RIIb, b1 and b2, are distinguished by the inclusion of a 19 amino acid in-frame insertion in the cytoplasmic tail
of b1 (Amigorena et al., 1992; Brooks et al., 1989; Firth et al., 2010). BLAST analysis shows that porcine transcript 1 is similar to human b1 and shares 77.4% homology with amino acid sequence of human b2, while porcine transcript 2 is the homologue of human b2, and shares 65.4% homology with amino acid sequence of human b2. The same as
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Fig. 3. Schematic representation of cDNA structure of five porcine Fc␥RIIb transcripts, which made up the different exons.
their human homologues, porcine transcript 1 also has a 19 amino acid in-frame insertion in the cytoplasmic domain whereas transcript 2 does not. It indicates that porcine transcript 1 may be recognised as the porcine Fc␥RIIb1 isoform, and transcript 2 as the porcine Fc␥RIIb2 isoform. There is only one alanine residue difference in the signal peptide region between porcine transcript 1 and transcript 4, or between transcript 2 and transcript 3. Moreover, transcript 3 and transcript 4 are also generated precisely by splicing style, the first two nucleotides of the consensus splicing donor (GT) and acceptor (AG), indicating that porcine transcript 3 or transcript 4 should belong to two new porcine Fc␥RIIb isoforms rather than mutants of transcript 2 and transcript 1. The identification of spliced form of the porcine transcript 5 is of particular interest, because this is the first time for the spliced form to be described in pigs rather than other animals and humans. This indicates that porcine transcript 5 is also a new novel porcine Fc␥RIIb isoform. It is clear that porcine Fc␥RIIb has at least five sub-isoforms now. The complexity of Fc␥RIIb-mediated immune response is demonstrated by the existence of Fc␥RIIb isoforms generated by alternative splicing. It is clear from earlier studies that both the murine and human b2 proteins undergo endocytosis upon binding ligand (Miettinen et al., 1989, 1992; Budde et al., 1994a; Van Den Herik-Oudijk et al., 1994). However, the endocytosis of immune complexs (ICs) mediated by b2 is not same as b1 in mice (Miettinen et al., 1989, 1992; Budde et al., 1994a). The b1 form uniquely prevents uptake of antigen-antibody complexes via clathrin-coated pits (Miettinen et al., 1992). It can be attributable to the increased affinity of the b1 insertion for cytoskeletal components, resulting from the association of b1 proteins with actin molecules and membrane phospholipids (Miettinen et al. 1992; Chen et al., 1999). Since immune complexes bound to b1 cannot be processed through the endocytic pathway, clonotypic expansion of Ag-specific T cells is prevented due to the lack of MHCII peptide presentation (Amigorena et al., 1992; Minskoff et al., 1998). In humans, bl and b2 display no notable difference in their signaling capabilities, as they both undergo
tyrosine phosphorylation, enhancing SHIP phosphorylation and downregulating cellular endocytosis (Joshi et al., 2006). However, there are conflicting reports with regard to the ability of b2 to become tyrosine-phosphorylated. In B cells, the human b2 ITIM tyrosine is not phosphorylated although its ability to downregulate antigen receptorinduced calcium mobilization is comparable to that of bl, which is tyrosine-phosphorylated (Budde et al., 1994b). Although the molecular details of this paradoxical observation are not fully understood, it is clear that human bl and b2 likely serve some non-overlapping functions in B cells. Of note, there are no studies to date comparing the functional capacity of porcine Fc␥RIIbl and b2. Likewise, it is not known whether both porcine Fc␥RIIbl and Fc␥RIIb2 are tyrosine-phosphorylated in B cells, macrophages and monocytes, and whether they function as inhibitory receptors during phagocytosis. In addition, in order to examine the tissue distribution of the porcine Fc␥RIIb in vivo, we surveyed amplicons by RT-PCR from porcine PBL cells and different tissue samples (muscle, thymus, lung, kidney, inguinal lymph node, spleen, tonsil, heart, liver) obtained from individual animals of a variety of porcine breeds, using the same primers as for PAM cells. The results showed that the porcine transcript 1 and transcript 3 were both detected and exhibited predominant expression in these tissues, PBL cells and PAM cells (data not shown). While the detection of these spliced variants is of great interest, it is beyond the scope of current research to elucidate mechanisms of the predominant expression pattern and the precise biological function. This topic is certainly a key area for future examination. Soluble Fc␥RIIb is first characterized in mice (Fridman and Golstein, 1974; Newport-Sautes et al., 1975). Soluble Fc␥RIIb is generated either by the cleavage of membranebound protein, or as soluble spliced variants lacking the exon coding for the transmembrane (TM) region (Fridman et al., 1993; Tartour et al., 1993; Galon et al., 1997). Membrane-cleaved soluble FcR can be produced by virtually any cell expressing the full-length protein, but it seems to be mainly produced by lymphocytes, at least in mice (Fridman et al., 1993). The TM negative form,
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Fig. 4. Flow cytometry analysis of expression of porcine Fc␥RIIb on the surface of COS-7 cells. (1) Representative histograms of the median fluorescence intensities (the positive population) of pcDNA-b1 transfected COS-7 cells surface-stained with Fc␥RIIb-specific pAb(A) or Fc␥RIIb-negative pAb(B). (2) The comparison of the MFI values of transfected COS-7 cells after surface staining with Fc␥RIIb-specific pAb and Fc␥RIIb-negative pAb. The data are means ± S.D. of three experiments, which are respectively 25.3 ±0.1 (A); 2.7 ± 0.2 (B); 17.7 ± 0.1 (C); 2.0 ± 0.2 (D); 20.0 ± 0.1 (E); 2.2 ± 0.2(F); 18.3 ±0.1 (G); 1.3 ± 0.1 (H); 14.4 ± 0.2 (I); 1.5 ±0.1 (J); 2.3 ± 0.2 (K); 1.5 ± 0.1 (L); 2.5 ± 0.1 (M); and 1.3 ± 0.1 (N).
however, is typically produced only by cells capable of expressing Fc␥RIIb2, such as T cells, macrophages and Langerhans. It is present in higher serum concentrations than the membrane-cleaved version (Fridman et al., 1993; Tartour et al., 1993). In cattle, Fc␥RIIb3, a soluble Fc␥RIIb, is characterized by the absence of a 40 amino acid region
concluding the predicted TM domain in b1 form. This transcript appears to have much more limited expression profile, which may indicate that the expression varies with the cellular activation-state (Firth et al., 2010). However, the transcript similar to bovine Fc␥RIIb3 variant has not been isolated in pig yet.
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Fig. 5. COS-7 cells transfected with porcine Fc␥RIIb genes bind IgG-sensitized erythrocytes (A) COS-7 cells transfected with pcDNA-b1. (B) COS-7 cells transfected with pcDNA-b2. (C) COS-7 cells transfected with pcDNA-b3. (D) COS-7 cells transfected with pcDNA-b4. (E) COS-7 cells transfected with pcDNA-b5. (F) COS-7 cells transfected with pcDNA3. (G) COS-7 cells.
The porcine alternatively spliced transcript 5 is particularly interesting. It is the first time that this form has been described in pigs rather than other animals and humans. Our results show the transcript 5 is unable to interact with immune complexes, despite its expression on the cell surface. One possible reason is that this protein lacks the EC2 domain, which is responsible for interacting with immune complexes and subsequent activations of innate immune cells (Hulett and Hogarth, 1994; Hulett et al., 1994, 1995). However, previous study reported that the EC1 domain of one Fc␥RIII receptor interacted with the EC2 domain of a successive Fc␥RIII molecule, which increased the accessibility of Fc␥RIII to the Fc portion of immune complexes and efficiently activated Fc␥RIII-inducing signals (Zhang et al., 2000). The function of transcript 5 in the porcine Fc␥RIIb-mediated immune responses needs further investigation. Generally, we have cloned five porcine Fc␥RIIb transcripts that are apparently generated according to the predicted splicing style. It is now clear that porcine Fc␥RIIb exists as at least five sub-isoforms, four of which can bind IgG. These isoforms may respectively modulate Fc␥RIIb-mediated immune responses in pigs. Hence, further functional characterization of Fc␥RIIb isoforms will provide new insight into better understanding of the
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